Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.     

辣根过氧化物酶在一种新型有机介质中的催化反应

选择合适的酶反应介质体系,是酶应用于有机合成的一个重要环节。利用适宜分子量的聚乙二醇（ＰＥＧ）可以将辣根过氧化物酶（ＨＲＰ）分散在甲苯中,摸索了ＨＲＰ在聚乙二醇（ＰＥＧ）－甲苯互溶体系反应的适宜条件,即ＰＥＧ／甲苯的比例、含水量、ｐＨ值、底物浓度等对酶活性影响,结果发现ＰＥＧ含量越低,含水量越高,酶的活力越高;酶在此体系中的最适ｐＨ值为７．０,最适过氧化氢浓度为２０ｍｍｏｌ／Ｌ,愈创木酚的浓度为０     

The Use of Salt Hydrates as Water buffers To Control Enzyme Activity in Organic Solvents

The conversion of o-diphenols to o-quinones was carried out in chloroform using a dry powder prepared from mushrooms as the catalyst. Several salt hydrates proved effective in supplying the small amount of water necessary for catalysis. The efficacy of the hydrates was related to their water activities and their use provided a convenient method for controlling water activity in nearly non-aqueous conditions.     

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.     

Protein adsorbents intended for use in aqueous two-phase systems

Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry to carry proteins away from the insoluble cell fragments generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume.     

低温光照下与黄瓜子叶叶绿素降低有关的酶促反应

黄瓜子叶在低温光照下处理时,随着时间的延长,Chl含量和CAT活性显著下降,POD和AsA-POD在开始24h活性亦有下降趋势,随后POD活性显著增加,而AsA-POD活性仅稍有提高。Chl含量的减少和CAT活性降低之间呈正相关,与POD活性的增加呈负相关。低温、光照处理的黄瓜子叶内Chl的降低可被亚胺环己酮(CHM)延缓,被AsA、Vit E、没食子酸丙酯(PG)和DCMU抑制,但被H_2O_2,乙醇酸钠和甲基紫精(MV)加速。     

Lipid peroxidative damage on cadmium exposure and alterations in antioxidantsystem in rat erythrocytes: A study with relation to time

Cadmium induced lipid peroxidation (LPO) and the activity of antioxidantenzymes after the administration of a single dose of CdCl 2 (0.4 mg kg body wt, ip) was studied in rat erythrocytes.Cd intoxication increased erythrocyte LPO along with a decrease insuperoxide dismutase (SOD) up to three days of Cd treatment. Thedecrease in erythrocyte catalase (CAT) activity was marked within9 h of Cd intoxication. After three days of Cd treatment, LPOdecreased towards normal, along with an increase in erythrocyteSOC and CAT activity. Blood glutathione (GSH) decreased significantlywithin 24 h of Cd treatment, followed by an increase towards normal.Erythrocyte glutathione S-transferase (GST) activity increased up to10 days of Cd intoxication, probably in an attempt to reduce Cd toxicity.Serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase(SALP) and serum bilirubin increased up to 10 days of Cd intoxication.Blood urea increased significantly up to three days, followed by a decreasetowards normal. The results show that Cd induced LPO was associated with adecrease in antioxidant enzymes and GSH in erythrocytes; as these antioxidantsincrease in erythrocytes with recovery from Cd intoxication, the Cd inducedLPO reversed towards normal. The increase in the SGPT, SALP and serum bilirubincorrelated with LPO. The results suggest that Cd intoxication induces oxidativestress and alters the antioxidant system, resulting in oxidative damage torat erythrocytes. © Rapid Science 1998     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.     

Differences in charge and kinetic properties of alcohol dehydrogenase 4 from C57BL/6 mice compared to other inbred strains are associated with a cysteine120 to arginine120 substitution

Alcohol dehydrogenase class IV (ADH4) participates in retinol metabolism and is expressed primarily in ocular, digestive, and reproductive tissues of the mouse. A naturally occurring genetic variant in C57BL/6J mice results in a faster migrating ADH4 enzyme during electrophoresis when compared to other non-C57BJ/6J strains. The C57BL/6 ADH4 gene coding sequence is found to have two nucleotide substitutions when compared to the gene from C3HeB/FeJ mice. The substitution in exon 5 encodes Arg120 instead of Cys120 in C57BL/6 ADH4 polypeptide; that would account for the protein electrophoretic phenotype. Arg120 is present in all published mammalian ADH4 sequences but is only in a limited number of mouse strains. The Arg120 residue is part of the outer loop of the substrate binding pocket and appears to have an effect on the affinity of the enzyme for several substrates.     

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: Evidence for genetic identity with stomach isozymes and localization ofAhd-4 on chromosome 11 near trembler

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those atAdh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C₂)] on chromosome 3. Three chromosome 11 markers, hemoglobin -chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established thatAhd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C₂ among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C₂ and of stomach and ocular AHD-4.This research was supported in part by the U.S. Department of Energy under Contract DE-ACO5-84OR214000 with Martin Marietta Energy Systems, Inc. (to R.A.P.).     

Synthesis of a Highly Substituted N-Linked Immobilized NAD Derivative Using a Rapid Solid-Phase Modular Approach: Suitability for Use with the Kinetic Locking-on Tactic for Bioaffinity Purification of NAD-Dependent Dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD⁺-dependent dehydrogenases. Specifically, the synthesis of highly substituted N⁶-linked immobilized NAD⁺ derivatives is described using a rapid solid-phase modular approach. Other modifications of the N⁶-linked immobilized NAD⁺ derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 Å) in an attempt to minimize nonbiospecific interactions. Analysis of the N⁶-linked NAD⁺ derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S⁶-linked immobilized NAD⁺ derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. -phenylalanine dehydrogenase ( -PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and -phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD⁺-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S⁶-linked immobilized cofactor derivatives than for the new N⁶-linked derivatives. In contrast, the NAD⁺-dependent phenylalanine dehydrogenase showed no affinity for the S⁶-linked immobilized NAD⁺ derivative, but was locked-on strongly to the N⁶-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N⁶-linked immobilized NADP⁺ derivative in the presence of -phenylalanine. This differential locking-on of NAD⁺-dependent dehydrogenases to N⁶-linked and S⁶-linked immobilized NAD⁺ derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD⁺ immobilized via N⁶-linkage as compared to thiol-linkage.     

Responses to chilling stress in French bean seedlings: antioxidant compounds

French bean (Phaseolus vulgaris L. cv. Contender) plants at five developmental stages (4, 8, 12, 16 and 20 d after sowing) were exposed to one of three treatments: 1 - 25 °C (control), 2 - exposure to chilling at 10 °C only for 2 d prior to sampling, and 3 - long-term exposure to chilling at 10 °C. Short- and long-term chilling decreased plant growth. Higher concentrations of ascorbate and glutathione were found in the chilling-treated plants throughout the different period of growth in comparison with those in the control plants. The activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase increased in the chilling-treated seedlings while activities of catalase and peroxidase and of β-carotene content decreased in young chilling-treated plants and slightly increased in older ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

Responses of antioxidative system to chilling stress in two rice cultivars differing in sensitivity

The responses of antioxidative system of rice to chilling were investigated in a tolerant cultivar, Xiangnuo-1, and a susceptible cultivar, IR-50. The electrolyte leakage and malondialdehyde content of Xiangnuo-1 were little affected by chilling treatment but those of IR-50 increased. Activities of suoperoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, and ascorbic acid content of Xiangnuo-1 were remained high, while those of IR-50 decreased under chilling. The results indicated that higher activities of defense enzymes and higher content of antioxidant under stress were associated with tolerance to chilling.     

Two alternative substrate paths for compound I formation and reduction in catalase-peroxidase KatG from Burkholderia pseudomallei

Five residues in the multifunctional catalase-peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase-specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8-A resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237-239. It is proposed that binding of substrate H(2)O(2) to Asp141 and Arg108 controls H(2)O(2) access to the heme active site, thereby modulating the catalase reaction.     

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

Biochemical genetic variants in mice selectively bred for sensitivity or resistance to ethanol-induced sedation

The distribution of biochemical genetic variants was examined among eight inbred strains of mice, which served as contributors to a heterogeneous stock of mice (HS), and in short-sleep (SS) and long-sleep (LS) mice, selectively bred from the HS stock for differential ethanol sensitivity. Fifteen loci for enzymes of alcohol and aldehyde metabolism, as well as 12 other biochemical loci, were investigated. Thirteen of these loci exhibited allelic variation between strains, of which six were separately fixed in the SS and LS mice. Comparisons of genetic similarity coefficients, based upon the distributions of allelic variants for the loci examined, with behavioural sensitivities (sleep-time) to an acute dose of ethanol for the inbred and selected strains of mice, indicated no correlations between these data. This suggests that this collective group of loci are not useful indicators of the genes selectively bred in the SS and LS strains, which are responsible for the differential sensitivities to acute doses of ethanol.     

Genetics and ontogeny of alcohol dehydrogenase isozymes in the mouse: Evidence for a cis-acting regulator gene (Adt-1) controlling C2 isozyme expression in reproductive tissues and close linkage of Adh-3 and Adt-1 on chromsome 3

An electrophoretic variant previously reported for the stomach isozyme of alcohol dehydrogenase (ADH-C2) in inbred strains of Mus musculus (Holmes, 1977) has been used to localize the gene encoding this enzyme (Adh-3) on chromosome 3 near Va (varitint) (9.6 ± 3.6% recombinants). Genetic variation of ADH-C2 activity in male and female reproductive tissues among inbred strains and Harwell linkage testing stocks was also observed. Reproductive tissue ADH-C2 phenotypes were inherited in a normal Mendelian fashion among F₂ progeny of an F₁ (LII × C57BL/Go) × C57BL/Go backcross as though controlled by a single cis-acting regulator locus (designated Adt-1) with two alleles: Adt-1 ^a (presence of ADH-C2) and Adt-1 ^b (absence or low activity of ADH-C2). No recombinants were observed among 73 progeny or among 13 inbred strains and six Harwell linkage testing stocks of mice, indicating that Adh-3 and Adt-1 are closely linked or identical genes. A single recombinant phenotype was observed in Peru-Coppock mice, suggesting that they are separate genes. Ontogenetic analyses demonstrated that ADH-B₂ is present throughout development from late fetal stages in stomach, liver, and kidney; similar results were found for ADH-C2 in developing kidney and stomach extracts, whereas ADH-A2 exhibited high activity in liver extracts after 3 weeks of age in both sexes and in male kidney extracts after 6 weeks.